Prophylaxis of allergic disease

ABSTRACT

The invention provides a method for preventing allergic disease in an individual susceptible to such disease, comprising administering an allergen to which the individual has not been sensitized previously. The allergen is administered in a dose and form effective to establish a stable population of allergen-specific T-helper-1-like memory lymphocytes capable of inhibiting activity or amplification of allergen-specific T-helper-2-like lymphocytes responsible for stimulating production of IgE antibodies specific for the allergen. Compositions for use in the method of the invention are also disclosed.

This application is the National Stage of International ApplicationPCT/AU94/00780, filed Dec. 19, 1994.

This invention relates to methods and compositions for the prophylaxisof allergic disease, and in particular to allergic disease triggered byenvironmental antigens or allergens.

The present inventor has extensively reviewed the literature relating toinduction of humoral and cellular immune responses to parenteral andenteral administration of allergens, and now proposes a novel andunexpected mechanism for inducing protective immunity against allergicdiseases, via selective stimulation of allergen-specific T-helper-1(TH-1) lymphocytes during early life.

For the purposes of this specification the following definitions areused:

allergen: any foreign antigen which stimulates allergic-type immuneresponses, characterised by activation of TH-2 lymphocytes andproduction of specific IgE antibody;

environmental allergen: any allergen found in the environment; suchallergens are usually, but not necessarily, naturally occurring;

sensitisation: “priming” of populations of T-cells to respondspecifically to subsequent challenge with the priming antigen orallergen; in the context of this specification, priming ofallergen-specific TH-2 cells;

desensitisation: therapeutic administration of allergen, or a derivativethereof, to allergen-reactive “allergic” individuals, with the aim ofselective suppression of the activity of allergen-specific T-cells, inparticular TH-2 cells, and/or other cell types recruited into theallergen-specific immune or allergic response.

BACKGROUND OF THE INVENTION

It was recognised early in this century that feeding experimentalanimals an antigen they had previously not encountered elicitedtransient symptoms of immediate hypersensitivity, which waned withcontinued food antigen exposure, to be replaced by a state ofantigen-specific unresponsiveness. The phenomenon is now known as OralTolerance, and has been shown to be preferentially directed againstIgE-mediated immediate hypersensitivity responses and delayed-typehypersensitivity responses (1). This form of tolerance can betransferred from animal to animal by T-cells secreting TH-1-likecytokines (2,3), and allergen specific T-cells secreting such cytokinesdevelop rapidly in the mesenteric lymph nodes during allergenfeeding.(10,12)

The inventor was the first to recognise the equivalent phenomenon in therespiratory tract, and has been investigating the underlying mechanismssince the early 1980s (4,5). The essential elements are identical:repeated inhalation of antigen aerosols elicits an initiallyheterogenous immune response which includes a component ofTH-2-dependent IgE production, but the latter eventually wanes in theface of repeated antigenic challenge, leaving only vestiges of specificIgG and IgA production. Animals passively exposed to antigen aerosols inthis fashion are unable to mount subsequent IgE responses to the sameantigen for the remainder of their lives, regardless of the route orintensity of challenge. As is the case with antigen feeding, the“tolerance” resulting from antigen inhalation is expressedpreferentially against IgE and delayed-type hypersensitivity, and ismediated by T-cells, including a population expressing CD8, whichsecrete TH-1-like cytokines(6), Additionally, the option for this formof “tolerance” induction appears open to the immune system only at oraround the time of initial allergen exposure—presensitised animals withstable on-going IgE responses are not “desensitised” by aerosolexposure(4,5).

These processes exhibit two further important features in common inexperimental animals. Firstly, sensitivity to tolerance induction isgenetically determined, and high sensitivity is co-inherited with thelow-IgE-responder-phenotype. Operationally, this manifests as arequirement for up to 10³ to 10⁴-fold more intense allergen exposure tosuccessfully tolerise high-IgE-responder rats and mice, compared totheir low-responder counterparts. However, it is clear that both highand low responders can ultimately be tolerised by either route, and theinherent sluggishness of these mechanisms in the high-IgE-responders canbe overcome by applying more intense allergen stimulation(4,5).

Secondly, the process functions poorly in the pre-weaning period(7), tothe extent that allergen exposure in the very early phase of infancy canprime for subsequent pathogenic T-cell reactivity, as opposed toinducing protective tolerance: this is consistent with the existence ofan early “window” of high risk for allergic sensitization, presumablydue to delayed postnatal maturation of one or more key elements ofmucosal immune function which are rate-limiting in the toleranceinduction process(7).

It is not clear to what extent mucosal allergen exposure via thegastrointestinal tract can suppress ongoing TH-2 responses inIgE-positive high-responder animals, but recent work employingintranasally administered allergen peptides encourages further pursuitof this approach in the context of desensitisation.

Initial exposure of humans to ubiquitous environmental allergens occursduring infancy or early childhood, and the notion that many of thetriggers for allergic disease in the adult are set during childhood isattracting increasing attention. In this context, there is a growingconsensus, based upon an expanding paediatric sero-epidemiologicalliterature, that high-level allergen. exposure during the first fewmonths of life predisposes to allergic sensitisation (7), whichmanifests in later childhood as TH-2-like reactivity. This implies that,as in experimental animals, transient maturational defect(s) in aspectsof immune function which are important for efficient “selection” forTH-1 reactivity to allergens encountered at mucosal surfaces may also becommon in newborn humans.

The present inventor has now recognized that the key element of therelevant human literature, however, is the characteristic biphasicnature of IgE responses to individual food and inhalant allergens whichcommonly occur during early childhood.

Thus, both normal children and those with a family history of atopicresponses typically develop serum IgE antibody responses against commonfood allergens during the first year of life, their magnitude andduration reflecting IgE-responder-phenotype(8). A comparable pattern isevident for IgE responses to inhalant allergens(8) (FIG. 1); however,the latter commence later in infancy, and take considerably longer toswitch off (“tolerise”) in the non-atopics. Furthermore, a much higherproportion of potential atopics maintain their serum IgE reactivity toinhalant allergens into later childhood than they do for foodallergens(8).

These differences in the kinetics and overall efficiency of “tolerance”induction to inhalant versus dietary antigens may derive directly fromthe differing levels of antigen exposure in the two organs: as T-cellsubset selection is “antigen driven”, the less intense stimulationprovided via low-level respiratory tract exposure may be expected toresult in a slower and ultimately less efficient process.

It is known that the magnitude and duration of IgE responses toparenteral antigenic challenge in experimental animals is regulated bycompeting signals from CD4⁺ T-cell subsets. In particular T-helper-2(TH-2) cells, which secrete interleukin-4 and interleukin-5, promoteIgE-B-cell switching, and TH-1 cells, which secrete interleukin-2 andinterferon-γ, inhibit TH-2 clonal expansion and hence limit the IgEresponse (9) The present inventor's review of a variety of data obtainedusing in vitro experimental systems employing human peripheral blood T-and B-cells indicates that an identical mechanism exists in man(10), andthis view is reinforced by the clear demonstration that both atopic andIgE-negative normal adults contain T-cells in peripheral blood which arereactive to the major inhalant allergens: in the atopic individuals,these cells appear to be predominantly of the TH-2 type, whereas innon-atopic individuals they appear to be mainly TH-1 (11) Considerabledebate surrounds the precise classification of these human T-cellsubsets relative to their murine counterparts, as respective cytokinepatterns are not identical in the two species; accordingly, currentopinion favours their classification in man as TH1-“like” andTH-2-“like” respectively.

Thus we suggest that in the non-atopic adult, each exposure to anenvironmental allergen would elicit a burst of TH-1-like cytokinerelease at sites of allergen presentation to the T-cell system, whichwould “protect” against the emergence of potentially pathogenicTH-2-like reactivity; each exposure event would additionally serve toconsolidate host-protective TH-1-like “memory”.

SUMMARY OF THE INVENTION

The present inventor now proposes that the T-lymphoid system in humansengages in active surveillance for environmental “allergens” throughoutlife, and that it is the nature of (as opposed to the mere presence of)allergen-specific T-cell responses in individuals that determineswhether they express the allergic (atopic) or immunologically normal(non-responder) phenotype. The inventor has recognised that selection ofthe appropriate T-cell population is an antigen-driven process whichoccurs during the early stages of immune responses in the naive(unsensitised) host. If selection favours the growth ofallergen-specific T-cells of the T-helper-1-like (TH-1)-like phenotypelow-grade non-pathogenic IgG and IgA responses ensue, whereas theemergence of TH-2-like cells can lead to IgE production and eosinophiliaand ultimately atopic disease. Additionally, TH-1-like cytokinesactively suppress the expansion of TH-2-like clones, and hence adominant, stable TH-1-like response to an allergen is proposed to beactively protective against the development of TH-2-like dependentallergic disease. With respect to T-cell responses to ubiquitousenvironmental allergens, the inventor's review of the recent paediatricliterature has identified early childhood as the life period duringwhich this selection normally occurs, and shows that the process cantake several years to complete. Once the significance of the selectionis appreciated, sufficient information is already known of how thisnatural selection process operates to contemplate controlling.it invivo, via deliberate administration of allergen(s) in a form adapted topreferentially stimulate the development of host-protective TH-1-likeimmunity.

Our data suggest that “bystander” cell populations, in particular CD8⁺and/or T_(γ/δ) cells, can actively assist the overall TH-1-likeselection process, and the term “TH-1-like immunity” in thisspecification is to be understood to include the contribution of thesecells.

According to a first aspect, the invention provides a method ofprevention of allergic disease in an individual susceptible to suchdisease, comprising the step of administering to a previouslyunsensitised individual a dose and form of allergen effective to induceestablishment of a stable population of allergen-specificT-helper-1-like memory lymphocytes, said lymphocytes being capable ofinhibiting activity or amplification of allergen-specificT-helper-2-like lymphocytes responsible for stimulating production ofIgE antibodies specific for said allergen.

Preferably the allergen is an environmental antigen, and may beadministered either singly or as a combination of two or more suchallergens. The allergen may be in its naturally-occurring form.Alternatively the allergen may be a protein prepared using recombinantDNA technology, or may be a synthetic peptide. The allergen may be inpurified form or may be impure or partially purified. The allergen mayrepresent either the whole allergen molecule, or may be a part thereof,for example including one or more epitopes. Allergens contemplated to besuitable for use in the invention include those from house dust mite,animal danders such as cat, dog or bird dander, cockroach, grass pollenssuch as those from ryegrass or alternaria, tree pollens such as thosefrom birch or cedar, feathers and moulds. The most suitable allergenswill depend on the geographical location. For example, birch and cedarpollens are a major cause of allergies in northern Europe and Japan, butare of minor importance in Australia.

For both aspects of the invention, the allergen may be administered bythe oral, intranasal, oronasal, rectal, intradermal, intramuscular orsubcutaneous route. The adjuvant is preferably a liposome or a microbialcell wall product.

The allergen may optionally be administered together with an adjuvant.Suitable adjuvants will be known to the person skilled in the art. Anadjuvant which selectively stimulates T-helper-1-like lymphocytes ispreferred.

The dose of allergen will generally be in the nanogram to milligramrange, depending on the allergen, the route of administration, andwhether or not an adjuvant is used. The person skilled in the art willreadily be able to determine the number and frequency of doses, usingwell-established principles. It is expected that for parenteraladministration the dose range will be of the order of micrograms, thatfor intranasal administration the dose range will be in the microgram tomilligram region, and that for oral or rectal administration the dosewill be in the milligram to gram range. It will be appreciated that thedose could vary depending on whether an adjuvant is used, and dependingon the nature of the adjuvant.

The method of the invention is suitable only for treatment ofindividuals who are not already allergic, i.e. hypersensitive, to theallergen being administered.

In general, the method is most suitable for treatment of childrenbetween 3 months and 7 years old, but is also applicable to individualsolder than 7 years. Preferably the immunization is administered tochildren not less than 6 months old, more preferably not less than 9months old.

Because in early childhood most individuals will not yet have beenexposed to sensitisation by environmental allergens, it is consideredthat this period provides the optimum opportunity to select forallergen-specific host-protective TH-1-like mediated immunity. It isespecially preferred that immunisation against airborne allergens, ie.allergens to which the individual is exposed by inhalation, be effectedduring early childhood.

According to one preferred embodiment of the invention, allergen isadministered orally or intranasally during early childhood.

According to a particularly preferred embodiment, which is considered toprovide selective induction of TH-1-like response to allergens withminimal stimulation of TH-2-like response, a mixture of two or moreallergens of the airborne type is administered parenterally togetherwith a TH-1-like selective adjuvant during early childhood.

According to a second aspect, the invention provides a sterilecomposition comprising an environmental allergen, together with anadjuvant capable of selectively stimulating T-helper-1-like lymphocytes,and optionally a pharmaceutically-acceptable carrier.

The allergen may be impure or purified, and may be of natural origin,produced by recombinant DNA technology, or synthetic.

Preferably the allergen is selected from the group which consists ofhouse dust mite, animal danders such as cat, dog or bird dander,cockroach, grass pollens such as those from ryegrass or alternaria, treepollens such as those from birch or cedar, feathers and moulds.

Preferably the composition is adapted for oral, intranasal, oronasal orrectal administration, but intradermal, subcutaneous or intramuscularadministration may also be used.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in detail, with reference followingnon-limiting examples, and to the accompanying drawings, in which

FIG. 1 illustrates post-natal serum IgE responses to environmentalallergens in normal and atopic children. Individual curves representresults of repeated serum samples from a single child; curves forindividual normal or atopic children fell within the regions shown. Thedata are derived from Reference 8. RAST represents radioallergoabsorbenttest for IgE; PRU represents paper radioimmimoabsorbent test for IgE(units/ml).

FIG. 2 shows selective suppression of IgE anti-ovalbumin (OVA) responsesby adoptive transfer of CD8⁺ lymphocytes from C57BI/6J mice rendered“tolerant” to OVA by repeated exposure. Open bars, IgE; hatched bars,IgG (asterisk, <controls; P<0.01).

FIG. 3 illustrates suppression of IgE response by γδ T cells. Open bars,IgE; hatched bars, IgG (asterisk, <controls; P<0.01).

FIG. 4 shows dose-response analysis of adoptive transfer of OVA-specifictolerance by positively selected γδ T cells from OVA-tolerant mice. Datashown are IgE titres from individual mice; the shaded area representsthe 95% confidence limits for the peak primary IgG response in normalanimals, and IgG titres in all animals in the experiment fell withinthis area. C*, comparable results obtained with untouched controls orrecipients of splenocytes from naive animals. Asterisk, <controls;P<0.01.

FIG. 5 illustrates the antigen specificity of γδ T cells in suppressionof primal IgE responses. Asterisk, <controls; P<0.01.

DETAILED DESCRIPTION OF THE INVENTION

Without wishing to be bound by any mechanism for the observed beneficialeffect, we propose that the “natural” mechanism for prevention ofallergic sensitization in humans is a cognate immunological process,which operates as follows:

(i) during early childhood, there is active immunological recognition ofthe major environmental allergens which are encountered, and thematuring immue system mounts low-grade, initially heterogenous T-cellresponses, comprising cross-competing TH-1-like and TH-2-likeallergen-specific clones;

(ii) during repeated rounds of restimulation via normal environmentalexposure, one of the competing T-cell phenotypes eventually becomesdominant in the response (typically TH-1-like in non-atopic normalsubjects), leading to the establishment of a stable pool of T-memorycells which “police” immune responses to the allergen throughout laterlife, preventing the emergence of TH-2-like clones reactive against thesame allergen.

With respect to inhalant allergens, this competition between co-existingantagonistic TH-1-like and TH-2-like T-cell populations appears tocontinue for a period of years during childhood, as these earlyallergen-specific IgE responses are often not terminated in non-atopicsuntil as late as age 5-7 years(8); (FIG. 1). Based on currentunderstanding of how T-cell reactivity develops, we consider thatresponses to inhalant allergens are “plastic” during this early period,and can be influenced toward either direction by exogenous factors. Inparticular, the known environmental risk factors for primary allergicsensitisation(7) must ultimately promote selection for TH-2-likereactivity. Additionally, the experimental literature indicates theexistence of a series of powerful mechanisms which can potentially pushthe equilibrium of the immune system towards selection forhost-protective anti-allergen responses, notably cytokines such asinterleukin-12 and interferon-α, produced by macrophages responding tocertain microbial stimuli(12), and interferon-γ produced byallergen-specific CD8⁺ T-cells(6), both of which select strongly forTH-1-like cells by inhibiting the expansion of TH-2-like cells.

The inventor has now recognised that the plasticity of these earlyallergen-specific immune responses, and their slow kinetics in vivo,provide potential opportunities for intervention. The period of earlychildhood, which has long been designated as the “window forsensitisation” to environmental allergens, can thus equally beconsidered as providing a “window of opportunity” for regulating thedevelopment of normal anti-allergen immunity in as yet unsensitisedchildren. Active intervention in the ongoing allergen-specific T-cellselection process which occurs during early childhood would optimiseselection for allergen-specific host-protective TH-1-like immunity: theslow overall kinetics of the natural immune response to inhalantallergens (see FIG. 1) suggests that such an approach would beparticularly applicable to prevention of sensitisation to suchallergens.

Firstly, it is proposed to accelerate and control the natural selectionprocess via either feeding or intranasal administration of allergen (orassociated peptides) during early childhood. This approach derives fromthe finding that even in atopics, the success with which nature“tolerises” emerging IgE responses to food allergens during infancy ismuch higher than is achieved with inhalant allergens, where overalllevels of allergenic stimulation are normally much lower. Thus enhancingthe overall level of inhalant allergen stimulation via the right routeat the right time may increase the overall efficiency of TH-1-likeselection.

It is emphasized that while this approach may appear superficiallysimilar to desensitisation strategies currently under development inmany laboratories, it is in fact precisely the opposite: the latter arebased upon usilencingf pathogenic TH-2-like cells in the sensitised hostwith pre-established TH-2-like memory, whereas the approach of thepresent invention is based on prevention of their emergence as a stablememory population in the first place.

A more direct strategy is suggested by recent developments in modernvaccine technology, which hold the promise of being able selectively toinduce TH-1-like responses to nominal antigens via appropriateparenteral immunisation, with minimal danger of stimulating parallelTH-2-like pathways. Thus deliberate parenteral vaccination with acocktail of the major inhalant allergens in appropriate TH-1-likeselective adjuvant at the appropriate time in childhood may provide asafe and reliable method to bolster populations of appropriate TH-1-likecells which are emerging as a result of natural mucosal stimulation,thus hastening their eventual dominance of allergen-specific T-memorypools. With respect to inhalant allergens, the finding that a highproportion of serum IgE in most atopics can be accounted for by arelatively small number of major environmental allergenspecificities(13) encourages the view that the relevant allergen(vaccine) cocktails may not necessarily be highly complex.

A preferred strategy is based on the use of adjuvants which:

a) stimulate the secretion of interleukin-12 and interferon-α bymacrophages, thus selecting for the growth of TH-1-like cells bymechanisms described in Reference 12; these adjuvants are likely to bederived from microbial products; and/or

b) selectively stimulate an initial burst of Class 1 MHC-restrictedimmunity against the administered allergen, in order to select for theensuing growth of allergen-specific TH-1-like cells by the mechanismwhich we have reported recently(6); a suitable adjuvant and deliverysystem for this purpose is expected to be various forms of liposomes, oran allergen-lipid conjugate, such as an iscom (an immune stimulatingcomplex comprising Quillaja saponis, cholesterol, phospholipid andantigen).

EXAMPLE 1 Selective Suppression of Primary Allergen-Specific IgEResponses by Pre-Induction of Class 1 MHC-Restricted Immunity

Mice were initially vaccinated against the allergen ovalbumin, using aprotocol designed to prime CD8⁺ T-cells. This protocol, which is knownper se, is based upon selective activation of CD8⁺ T-cells by initialpriming with spleen cells which have been cytoplasmically “loaded” withsoluble ovalbumin by osmotic shock. Our preliminary results indicatethat the vaccinated mice are unable to mount subsequent high titreprimary antigenovalbumin IgE response to parenteral challenge withovalbumin, but are able to make normal IgG responses. This indicatesthat the initial vaccination selectively suppressed the TH-2-likecomponent of the anti-ovalbumin response of these mice, whereasTH-1-like dependent IgG production proceeded normally. This resultclearly supports the principles underlying the proposed vaccinationstrategy, and further experiments involving alternative vaccinationprotocols designed to achieve the same end result are in progress.

EXAMPLE 2 Use of Liposomes as Adjuvants

It has been suggested that liposomes can be used as vehicles fordelivery of antigens in order to generate anti-viral immunity, inparticular immunity based upon the generation of mixed “memory” inantigen-specific CD8⁺ and CD4⁺ TH-1-like viral antigen-specific T-cellpopulations. This strategy is being used for generation ofallergen-specific CD8⁺ TH-1-like immunity in mice, which is expected tobe highly protective against the subsequent development of TH-2-likedependent IgE responses to these allergens. Various formulations ofliposomes and allergens are being tested.

EXAMPLE 3 Microbial Cell Wall Products as Selected TH-1-Like Adjuvants

We and others have demonstrated that parenteral challenge of mice withcertain microbial cell wall-derived adjuvants selectively suppressesTH-2-like dependent IgE responses, while stimulating TH-1-like dependentIgG responses. The most commonly available adjuvants, such as Freund'scomplete adjuvant, are not suitable for human use, is as they coursetissue necrosis at the injection site. Cell wall extracts from a widevariety of bacterial strains are being tested in order to identifypreparations which are both non-toxic and TH-2-like suppressive, using ascreening protocol based on co-injection of extract together withovalbumin into mice, and measuring the subsequent ovalbumin-specific IgEand IgG responses. A variety of cell wall-derived adjuvants fromMycobacterium tuberculosis, such as muramyl dipeptide, have beenintensively investigated as potential adjuvants for human use, and it iscontemplated that these may be useful for the purposes of thisinvention.

EXAMPLE 4 Use of a Modified Allergen as an IgE “Tolerogen”

Some recent publications have indicated that protein antigensartificially modified by the addition of conjugated lipid “tails” elicitClass I MHC-restricted immune responses, whereas the native proteinsstimulate an exclusively Class II MCH-restricted response. As discussedabove, according to the principles underlying the present invention sucha modified antigen should also selected for TH-1-like immunity to theantigen, thus inhibiting the development of a TH-2-like dependent IgEresponse. Mice which had not been previously exposed to the allergenovalbumin (OVA) were parenterally immunised and subsequently challengedwith either native ovalbumin, or ovalbumin which had been structurallymodified by conjugation with the lipid dodecenoic acid. Mice wereinitially primed with either native ovalbumin as a control, or with thelipid conjugate (Dodec-OVA) on Day 0, bled on Days 14 and 20, challengedwith the same preparations respectively on Day 25, and bled again on Day39. Serum titres of anti-ovalbumin IgE antibody were measured, and theresults, presented as group median passive cutaneous anaphylaxis units,are shown in Table 1.

TABLE 1 Ovalbumin Dodec-OVA Day 14 1280 <40 Day 20 1280 <40 Day 39 1280<40

These results clearly show that priming with a modified antigen whichselects for TH-1-like immunity does indeed prevent subsequentdevelopment of a TH-2-like dependent IgE response.

EXAMPLE 5 γS T Cells Regulate IgE Responses to Inhale Allergen

We have previously shown that either oral or intranasal administrationof allergen to animals which have not been previously exposed to thisallergen can confer active protection against production ofallergen-specific IgE by induction of a state of allergen-specificimmunity which results in TH-1-like cytokine responses each time theallergen is encountered (6). Our earlier publication identified thecellular sources of these cytokines as being both Class I MHC-restrictedCD8⁺ T Cells and Class II MHC-restricted CD4⁺ T cells.

We have now shown that allergen-specific T γδ cells provide a furthersource of TH-1-like cytokines in these responses; similar results havebeen obtained in both mice and rats.

C57BI/6J mice were exposed daily for 10 days to aerosolized OVA inphosphate-buffered saline and once weekly thereafter until used asdescribed in our earlier work (8). Intraperitoneal (ip) challenge of asubgroup of these animals with 10 μg of OVA in 4.0 mg of aluminiumhydroxide (AH) adjuvant revealed normal primary IgG responses butvirtually complete suppression of parallel IgE responses, asdemonstrated in our earlier studies (8). Splenocytes were prepared fromother (unchallenged) “tolerant” animals and divided into three samples.The first sample was left unfractionated, the second was negativelydepleted of CD8⁺ cells by cytometry, and CD8⁺ cells were purified fromthe third using positive selection by cytometry (Epics Elite, CoulterElectronics); the CD8⁺ antibody used was from the 53-6.72 clone (14) andthe cytometry methodology used was as previously described (7). The CD8⁺population was more than 99.5% pure, and the CD8⁻ population containedless than 0.4% of contaminating CD8⁺ cells. Immediately after ipinjection of these cell populations, animals were immunised ip with 10μg of OVA in 4.0 mg of AH adjuvant, and bled at Days 14 and 21.

IgG subclasses were measured by an enzyme-linked immunosorbent assay(ELISA) with anti-IgG subclass antibodies (Southern Biotechnology).Splenocytes were prepared as previously described and passed throughnylon wool to remove adherent cells, thus yielding˜85% T cells. Negativeselection of αβ T cells was performed by flow cytometry with H57-597.19(anti-αβ TCR) (15). γδ T cells constitute approximately 30% of theremaining cells; hence 1×10⁵ splenocytes will contain 3×10⁴ γδ T cells.

Adoptive transfer of 10⁶ unfractionated splenocytes from the tolerisedmice inhibited IgE, but not IgG, antibody responses to ovalbumin in therecipient animals. These results are illustrated in FIG. 2.

The data shown are the mean±SD (n=5 to 10 per group) at Day 21 (peakprimary Ig response) and indicate reciprocal log2 (IgE and IgG) anti-OVAtitres as determined by standard methods (7). Data from Day 14 did notalter the interpretation of the results of these experiments.

The magnitude of the overall IgG anti-OVA response did not changesignificantly in mice pretreated with OVA aerosol. However, analysis ofindividual IgG subclasses by ELISA with subclass-specific anti-IgGantibodies (Southern Biotechnology) showed that suppression of the IgEresponse was accompanied by decreased IgG₁ reactivity and a compensatoryrise in IgG_(2a), whereas IgG_(2b) and IgG₃ responsiveness wasessentially unchanged.

EXAMPLE 6 Dose-Response Relationships for γδ T Cells

Depletion of γδ⁺ T cells abolished the capacity of splenocytes tosuppress the IgG response. There are approximately 3×10⁴ γδ T cells per10⁶ splenocytes. When this number of γδ T cells purified to >98.5% bypositive selection was transferred to recipient animals, the degree ofsuppression of the IgE response was comparable to that seen in animalsreceiving 10⁶ unfractionated cells. This is illustrated in FIG. 3.Spleen cells from tolerized animals were negatively depleted of γδ Tcells with the antibody GL3 (16). GL3⁺ (γδ⁺) cells were prepared bypositive selection. Adoptive transfer, ip antigen challenge, anddetermination of primary IgE and IgG responses were performed as above.These results are shown in FIG. 3.

Dose-response experiments showed that as few of 5×10² positivelyselected γδ T cells are sufficient for suppression of the IgE componentof anti-OVA response. γδ T cells were prepared by negative selectionfrom OVA-tolerant donors. Splenocytes were prepared as above and passedthrough nylon wool to remove adherent cells, yielding approximately 85%T cells. Negative selection of αβ cells was achieved by flow cytometrywith H57-57.19 (anti-αβ TCR) (17). γδ T cells constitute approximately30% of the remaining cells, so that 1×10⁵ splenocytes will contain 3×10⁴γδ cells. These γδ cells yielded a suppression of the Igz responsecomparable to that achieved with positively selected cells, as shown inFIG. 4. We have previously shown that adoptive transfer of splenocytesdepleted of αβ⁺ T cells from OVA-tolerance rats was capable of mediatingantigen-specific tolerant in the IgE isotype (17).

EXAMPLE 7 Antigen Specificity of γδ T Cell-Mediated Suppression

In order to test for the antigen specificity of the γδ T cell-mediatedsuppressive response, unfractionated splenocytes or purified γδ T cellswere transferred from OVA-tolerant mice to groups of syngerneicrecipients, which were then challenged with OVA or an unrelated antigen,Der P1 from the house dust mite. The transferred cells suppressedprimary anti-OVA responses, but did not affect corresponding anti-Der P1responses. Unfractionated or positively selected γδ T cells fromOVA-tolerant rats were transferred, and the recipients were challengedwith OVA or Der P1. The results are shown in FIG. 5. Antigen specificitywas observed in this system, even at 50-fold higher cell dosages.

EXAMPLE 8 Cytokine Production in Mice Tolerised to OVA

Splenocytes from mice tolerized to OVA were challenged In vitro with 100μg/ml OVA, and supernatants of these cells were harvested after 24 hrsfor assessment of cytokine production. Splenocytes were depletedof >99.5% CD4⁺, CD8⁺ αβ⁺ or γδ⁺ T cells by negative selection, usingflow cytometry. Cells were cultured at 2×10⁵ per microplate well in RPMImedium containing 10⁻⁵ M 2-mercaptoethanol plus antibiotics,supplemented with 1 to 10% foetal calf serum, and stimulated with 100μg/ml OVA. Supernatants were harvested after 24 hrs, and frozen at −20°C. prior to assay. IL-2 secretion was measured using a standard CTLLassay (6), and IFN-γ and transforming growth factor β1. TCF-β1 weredetermined by ELISA (Pharmingen and Genzyme respectively). The cells didnot respond to an irrelevant control antigen, and cells from unimmunizedcontrol animals did not secrete detectable levels of cytokines inresponse to OVA.

Unfractionated splenocytes from tolerant animals secreted high levels ofinterferon-γ (IFN-γ) in response to specific antigen, and this secretoryresponse was markedly reduced by depletion of CD8⁺ cells, but not CD4⁺cells. Depletion of CD8⁺ cells markedly enhanced the OVA-specificinterleukin-2 (IL-2) response. These results are summarized in Table 2,which shows mean ±standard deviation (SD) for replicate 24 hr culturesupernatants.

TABLE 2 Cytokine secretion IFN-γ IL-2 TGF-β1 Cells (ng/ml) (U/ml)(ng/ml) Unfractionated 226.5 ± 7.8  2.7 ± 0.3 1.59 ± 0.15 CD4⁻ 245.0 ±28.3 1.8 ± 1.1 1.84 ± 0.73 CD8⁻  63.5 ± 12.0 9.3 ± 0.7 2.01 ± 0.57 αβ⁻147.5 ± 21.9 2.6 ± 1.9 1.65 ± 0.39 γδ⁻ 90.0 ± 2.5 8.7 ± 1.2 1.98 ± 0.24Non-T cells ND ND 0.45 ± 0.09 (CD3⁻) ND: not determined.

TGF-β1 was also measured, since it has been suggested to play animportant role in CD8⁺ T cell-mediated tolerance to orally-administeredantigens (20). However, as shown in Table 1, TGF-β1 was in fact found tobe produced in similar amounts by all T cell subsets after antigenicstimulation of tolerant animals, regardless of the potency of thesubsets in transfer of tolerance. This suggests that TGF-β1 does notplay a central role.

EXAMPLE 9 Proliferative Response to OVA of Splenic T Cells fromAerosol-Exposed Mice

We also examined the ability of splenic T cells to proliferate in vivoin response to OVA, following prior exposures of mice to OVA aerosols.The negative selection of αβT cells was performed as described above,and proliferation measured after stimulation with 100 μg/ml of OVA.Results, presented as mean ±SD of replicate cultures measured after 96hrs incorporation of [³H]-thymidine, are shown in Table 3. The subsetscontained not more than 0.5% contaminant cells.

Proliferation Cell population ([³H]DNA Synthesis) Undepleted 5,232 ± 75αβ⁻    699 ± 32 γδ⁻ 13,255 ± 563

Cells from OVA-exposed animals did not proliferate in response to anunrelated control antigen, and normal cells did not proliferate in thepresence of OVA. A moderate proliferative response to antigen wasconsistently seen in unfractionated splenocytes; this was abrogated bydepletion of αβ T cells, and enhanced by depletion of the γδ subset. Asshown in Table 2, the latter procedure was also accompanied by a largeincrease IL-2 production. This suggests inhibition of αβ T cellproliferation by the γδ population, which is consistent with thereported effects of in vivo γδ T cell depletion (21).

We conclude that the effector cells mediating the selective suppressionof IgE responses in this model in mice are CD4⁻ CD8⁺ γδ⁺ T cells whichare specific for OVA. From Table 1, it appears that these cells secreteinterferon-γ in response to OVA, which is consistent with reports of γδcell responses to stimulation with microbial antigens (22). They mayalso trigger interferon-γ release from other cell populations, such asnatural killer cells, which are CD4⁻, or from CD8⁺ αβ T cells, both ofwhich can be potent sources of interferon-γ (22). Thus the T cellresponse to inhaled OVA in tolerised mice displays a TH-1-like profile,which is consistent with the pattern of selective suppression ofspecific IgE and IgG₁ production and concomitantly enhanced IgG_(2a)secretion observed in these animals. Our results suggest an importantrole for antigen-specific γδ T cells in the maintenance of immunologicalhomeostasis in the lung and airways by selective suppression ofpotentially pathogenic TH-2-like dependent IgE responses, whilepreserving the host's capacity to produce specific IgG antibody.

EXAMPLE 10 Induction of Class I MHC-Restricted Immunity Against OVA ViaTransfection

In order to gain more definitive evidence that immunisation whichresults in a selective boosting of CD8-mediated responses againstallergens can confer protection against development of TH-2-likereactivity, we have primed for anti-OVA immunity via inoculation of micewith a cell line which expresses Class I but not Class II MHC responses,and which has been transfected with the gene encoding OVA. These cellsproduced OVA intracellularly, resulting in introduction of OVA intocytoplasmic Class I antigen-processing pathways, which are generallyinaccessible to exogenous proteins. The transfected cells thereforepresent complexes of “processed” OVA or their surface in conjuntion withClass I MHC antigens. This primes Class I MHC-restricted CD8⁺ T cells inthe recipient animals for anti-OVA immunity. Primed animals and controlswere then challenged parenterally with native OVA and bled 14 and 30days later for determination of IgE anti-OVA titres. The results,presented as group medians in passive cutaneous anaphylaxis units, areshown in Table 4.

TABLE 4 Control Primed Day 14 640 40 Day 30 640 40

OVA-specific reactivity was in fact demonstrated. These results areconsistent with our overall postulate.

It will be apparent to the person skilled in the art that while theinvention has been described in some detail for the purposes of clarityand understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and areincorporated herein by this reference.

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What is claimed is:
 1. A method for the prophylactic treatment ofallergic disease triggered by an environmental allergen in a humanindividual susceptible to such disease, comprising the step ofadministering to an individual who has not been sensitized by saidenvironmental allergen a dose and form of said environmental allergeneffective to induce establishment of a stable population ofallergen-specific T-helper-1-like memory lymphocytes, said lymphocytesbeing capable of inhibiting activity or amplification ofallergen-specific T-helper-2-like lymphocytes responsible forstimulating production of IgE antibodies specific for said environmentalallergen.
 2. A method according to claim 1, wherein the individual is 3months to 7 yers old.
 3. A method according to claim 2, wherein theindividual is 6 months to 7 years old.
 4. A method according to claim 3,wherein the individual is 9 months to 7 years old.
 5. A method accordingto claim 1, wherein the allergen is administered together with animmunological adjuvant.
 6. A method according to claim 5, wherein theadjuvant is one which selectively stimulates T-helper-1-likelymphocytes.
 7. A method according to claim 1, wherein the environmentalallergen is selected from the group consisting of insect allergens,pollens, animal danders, bird danders, feathers, and moulds.
 8. A methodaccording to claim 1, wherein the environmental allergen is an inhaledallergen.
 9. A method according to claim 1, wherein two or moreallergens are administered for the prophylactic treatment of allergicdiseases triggered by said two or more allergens.
 10. A method accordingto claim 1, wherein the environmental allergen is of biological origin.11. A method according to claim 1, wherein the allergen is administeredby the oral, intranasal, oronasal, rectal, intradermal, intramuscular orsubcutaneous route.
 12. A method according to claim 11, wherein theallergen is administered orally or intranasally.